The presence of hepatitis B virus (HBV) DNA was investigated in the sera from 25 Japanese patients with hepatocellular carcinoma (HCC) using the polymerase chain reaction (PCR) method. Two stages of PCR amplifications (30 cycles each) for three different gene segments of HBV DNA, the PreC/C, S and X genes, were performed using the outer and inner primer pairs. The three target sequences were amplified separately (standard PCR) or simultaneously ("multi-target" PCR). Fifteen of the 16 hepatitis B surface antigen (HBsAg)-negative sera were negative for all three gene segments; the remaining one serum was positive only for the PreC/C gene segment. By contrast, 8 of 9 HBsAg-positive sera were positive for all three gene segments; the remaining one serum could not be determined to be positive or negative. Additional serologic studies (e.g., HBeAg, anti-HBc) were also conducted.